Immunocytochemical and pharmacological studies have provided evidence that calcium activated thiol proteases (calpains) plays am important role in neuropathology. Partial digestion of an endogenous substrate for these enzymes occurs in several pathogenic circumstances, in some cases days before the onset of overt signs of cellular degeneration, and a drug that inhibits calpain blocks the development of a=pathology in two paradigms in which it has been tested. In light of these results, it is now reasonable to begin exploring the possibility that calpain, acting in concert with other factors, also contributes to age-related changes in the brain. Four projects of this type constitute the present proposal. Experiment 1 will test if the concentration of a breakdown product that results from the digestion of spectrin by calpain increases in the aged mouse brain. Pilot data point to this conclusion; if confirmed, this would provide indirect evidence for greater calpain activity, with aging. Experiment 2 will use pharmacological stimulation of NMDA receptors and hypoxia to determine if the aged brain is more vulnerable to pathogenic conditions and if this is associated with an enhanced activation of calpain. Preliminary work suggests that episodes of hypoxia too short to cause aged slices. The proposed studies will extend this project and test if the aged slices are also more vulnerable to NMDA receptor stimulation. Assays of spectrin breakdown and physiological tests of the protective effects of calpain inhibitors will be used to assess the likelihood that excessive stimulation of the protease contributes to pathological responses in the aged brain. These studies will include recently introduced calpain inhibitors that appear to be more potent and selective. Experiment 3 will examine the possibility that the increased degradation of calpain substrates found in pathogenesis and possibly aging is due in part to a facilitation of the interactions between substrates and protease. Membranes will be isolated from the brain of gerbils after transient ischemia and from different regions of the aged rat brain and proteolysis assessed following an incubation with exogenous calpain. Experiment 4 will determine if various components of the amyloid precursor protein are substrates for calpain and if pathogenic manipulations trigger the partial digestion of the protein. These experiments should provide evidence needed to evaluate the possibility that excessive activation of calpain contributes to amyloid formation in aging.